Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver. Purification, characterization and subcellular localization

1. Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver was purified to homogeneity. 2. The purified enzyme moves as a single electrophoretic band at pH 8.3 in 7.5% acrylamide and was coincident with the enzyme activity. 3. Gel filtration on Sephadex G-200 gave an apparent molecular weight of 110,000 with two apparent identical subunits of 54,000-56,000 as determined by sodium dodecyl sulphate gel electrophoresis. 4. The maximum of enzyme activity was obtained in the presence of 3-5 mM ZnCl2 at pH 6-6.2, however, higher concentrations of metal are inhibitory. The enzyme hydrolyses p-nitrophenylphosphate, o-carboxyphenylphosphate and phenylphosphate, was insensitive to NaF and was inhibited by phosphate and ATP. The Km for p-nitrophenylphosphate was 0.28 x 10(-3)M at pH 6 in 50 mM sodium acetate/100 mM NaCl. 5. Phosphate is a competitive inhibitor (Ki = 0.5 x 10(-3)M) whereas ATP seems to be a non-competitive inhibitor (Ki = 0.35 x 10(-3)M). The isoelectric point determined by isoelectric focusing on polyacrylamide gel is 7.5. 6. Cell fractionation studies indicate that the Zn2+-dependent acid p-nitrophenylphosphatase of chicken liver is a soluble enzyme form.     

Purification,characterization and developmental expression of pig liver PSP

We have isolated a perchloric acid-soluble protein designated as PL-PSP from the post-mitochondria supernatant fraction of pig liver. It is soluble in 5% perchloric acid and purified by ammonium sulfate fractionation and CM-Sephadex chromatography. The PL-PSP showed approximately 80–90% homology with PSP isolated from rat liver (RL-PSP) with its partial amino acid sequences. The protein has a molecular mass of approximately 14 kDa which was slightly higher than that of RL-PSP. It inhibited protein synthesis in a rabbit reticulocyte lysate system. The expression of PL-PSP was predominant in liver, kidney and duodenum, and was also expressed in stomach, lung and brain. PL-PSP expression in liver increased from the 1st day to the 1st month. Thus, our findings are the first report on the presence of a PSP in porcine tissues which may be involved in the regulation of cellular growth and differentiation.     

Purification and characterization of dihydropyrimidine dehydrogenase from pig liver

Dihydropyrimidine dehydrogenase was isolated from cytosolic pig liver extracts and purified 3100-fold to apparent homogeneity. Purification made use of ammonium sulfate fractionation, precipitation with acetic acid and chromatography on DEAE-cellulose and 2',5'-ADP-Sepharose with 28% recovery of total activity. The native enzyme has a molecular mass of 206 kDa and is apparently composed of two similar, if not identical, subunits. Proteolytic cleavage reveals two fragments with apparent molecular masses of 92 kDa and 12 kDa. The C-terminal 12-kDa fragment seems to be extremely hydrophobic. The enzyme contains tightly associated compounds including four flavin nucleotide molecules and 32 iron atoms/206-kDa molecule. The iron atoms are probably present in iron-sulfur centers. The flavins released from the enzyme were identified as FAD and FMN in equal amounts. An isoelectric point of 4.65 was determined for the dehydrogenase. Apparent kinetic parameters were obtained for the substrates thymine, uracil, 5-aminouracil, 5-fluorouracil and NADPH.     

Purification, characterization, and immunocytochemical localization of the major basic protein of pig blastocysts

The major basic protein (BP) synthesized and secreted by elongating pig blastocysts was purified from medium of Day 14-17 conceptus cultures. Sequential ion-exchange and gel-filtration chromatographies resulted in isolation of BP as a single polypeptide of Mr = 43,100 or 42,800 under denaturing or native conditions, respectively. BP was found to be a glycoprotein by incorporation of [3H] glucosamine and susceptibility to N-glycopeptidase F. Two BP polypeptides were produced by N-glycopeptidase F (Mr = 39,800 and 36,300). Antiserum to BP immunoprecipitated radiolabeled BP from blastocyst culture medium. BP was not detected in medium from 1-2 mm diameter spherical (Day 10) blastocysts but was found in medium from 3-5 mm spherical (Day 10) and filamentous (less than 50 cm, Day 12) conceptuses, suggesting that BP synthesis and secretion began at the initiation of trophoblast expansion. With immunocytochemical procedures, BP was located in the apical cytoplasm of trophectoderm cells of Day 11 expanding (5-7 and 10-20 mm) blastocysts. These results suggest that trophoblast epithelium secrete BP apically toward the uterine lumen and that BP may play a role in maternal-fetal interactions during the peri-implantation period.     

Purification and characterization of glutathione S-transferases from guinea pig liver

Four types of glutathione S-transferase were purified to homogeneity from guinea pig liver by DEAE-cellulose, Sephadex G-75, CM-cellulose, and affinity chromatography. These isozymes were named a, b, c, and d based on the reverse order of elution from a CM-cellulose column, and had specific activities of 89.6, 92.2, 99.0, and 44.0 units/mg, respectively, when assayed with 1 mM each of 1-chloro-2,4-dinitrobenzene and reduced glutathione. All four transferases of guinea pig liver were homodimers. The transferases b, c, and d had a similar molecular weight of 50,000 and their subunit sizes were 25,000, but the corresponding values for transferase a were 45,000 and 23,500, respectively. Transferase a was notably different in the activities towards organic hydroperoxides and 1,2-dichloro-4-nitrobenzene from the other isozymes. Transferases a and b, the major forms in guinea pig liver, were studied with respect to their biochemical properties, including kinetic parameters, absorption and fluorescence spectra, and bilirubin binding. Glutathione peroxidase activity of the transferase a was about 100 times higher than that of other isozymes. In guinea pig liver, it is estimated that transferase a is the major glutathione peroxidase, accounting for about 75% of the total organic hydroperoxide reduction.     

Purification and characterization of trimming glucosidase I from pig liver

Trimming glucosidase I has been purified about 400-fold from pig liver crude microsomes by fractional salt/detergent extraction, affinity chromatography and poly(ethylene glycol) precipitation. The purified enzyme has an apparent molecular mass of 85 kDa, and is an N-glycoprotein as shown by its binding to concanavalin A-Sepharose and its susceptibility to endo-beta-N-acetylglucosaminidase (endo H). The native form of glucosidase I is unusually resistant to non-specific proteolysis. The enzyme can, however, be cleaved at high, that is equimolar, concentrations of trypsin into a defined and enzymatically active mixture of protein fragments with molecular mass of 69 kDa, 45 kDa and 29 kDa, indicating that it is composed of distinct protein domains. The two larger tryptic fragments can be converted by endo H to 66 kDa and 42 kDa polypeptides, suggesting that glucosidase I contains one N-linked high-mannose sugar chain. Purified pig liver glucosidase I hydrolyzes specifically the terminal alpha 1-2-linked glucose residue from natural Glc3-Man9-GlcNAc2, but is inactive towards Glc2-Man9-GlcNAc2 or nitrophenyl-/methyl-umbelliferyl-alpha-glucosides. The enzyme displays a pH optimum close to 6.4, does not require metal ions for activity and is strongly inhibited by 1-deoxynojirimycin (Ki approximately 2.1 microM), N,N-dimethyl-1-deoxynojirimycin (Ki approximately 0.5 microM) and N-(5-carboxypentyl)-1-deoxynojirimycin (Ki approximately 0.45 microM), thus closely resembling calf liver and yeast glucosidase I. Polyclonal antibodies raised against denatured pig liver glucosidase I, were found to recognize specifically the 85 kDa enzyme protein in Western blots of crude pig liver microsomes. This antibody also detected proteins of similar size in crude microsomal preparations from calf and human liver, calf kidney and intestine, indicating that the enzymes from these cells have in common one or more antigenic determinants. The antibody failed to cross-react with the enzyme from chicken liver, yeast and Volvox carteri under similar experimental conditions, pointing to a lack of sufficient similarity to convey cross-reactivity.     

Purification and characterization of monolysocardiolipin acyltransferase from pig liver mitochondria

In mammalian tissues cardiolipin is rapidly remodeled by monolysocardiolipin acyltransferase subsequent to its de novo biosynthesis (Ma, B. J., Taylor, W. A, Dolinsky, V. W., and Hatch, G. M. (1999) J. Lipid Res. 40, 1837-1845). We report here the purification and characterization of a monolysocardiolipin acyltransferase activity from pig liver mitochondria. Monolysocardiolipin acyltransferase activity was purified over 1000-fold by butanol extraction, hydroxyapatite chromatography, and preparative SDS-PAGE. The purified 74-kDa protein catalyzed acylation of monolysocardiolipin to cardiolipin with [(14)C]linoleoyl coenzyme A. Photoaffinity labeling of the protein with 12-[(4-[(125)I]azidosalicyl)amino]dodecanoyl coenzyme A indicated coenzyme A was bound at its active site and photoaffinity cross-linking of 12-[(4-azidosalicyl)amino]dodecanoyl coenzyme A to the enzyme inhibited enzyme activity. Enzyme activity was optimum at pH 7.0, and the enzyme did not utilize other lysophospholipids as substrate. The purified enzyme was heat-labile and exhibited an isoelectric point of pH 5.4. To determine the enzymes kinetic mechanism the effect of varying concentrations of linoleoyl coenzyme A and monolysocardiolipin on initial velocity were determined. Double-reciprocal plots revealed parallel lines consistent with a ping pong kinetic mechanism. When the enzyme was incubated in the absence of monolysocardiolipin, coenzyme A was produced from linoleoyl coenzyme A at a rate consistent with the formation of an enzyme-linoleate intermediate. The true K(m) value for linoleoyl coenzyme A and true K(m) value for monolysocardiolipin were 100 and 44 microM, respectively. The calculated V(max) was 6802 pmol/min per mg of protein. A polyclonal antibody, raised in rabbits to the purified protein, cross-reacted with the protein in crude pig liver mitochondrial fractions. In liver mitochondria prepared from thyroxine-treated rats, the level of the protein was elevated compared with euthyroid controls indicating that expression of monolysocardiolipin acyltransferase is regulated by thyroid hormone. The study represents the first purification and characterization of a monolysocardiolipin acyltransferase activity from any organism.     

Purification and characterization of guinea pig liver morphine 6-dehydrogenase

Morphine 6-dehydrogenase, which catalyzes the dehydrogenation of morphine to morphinone, has been purified about 440-fold from the soluble fraction of guinea pig liver with a yield of 38%. The purified enzyme was a homogeneous protein on polyacrylamide gel disc electrophoresis and isoelectric focusing. The molecular weight and isoelectric point of the enzyme were 29,000 and 7.6, respectively. The enzyme utilizes both NAD and NADP as a cofactor, and the Km values were 0.12 mM for NAD and 0.42 mM for NADP. The Vmax values for morphine were 588 milliunits/mg of protein (with NAD) and 1600 milliunits/mg of protein (with NADP). The Km values for morphine were 0.12 mM (with NAD) and 0.49 mM (with NADP). The enzyme also exhibited activity for morphine-related compounds: nalorphine, normorphine, codeine, and ethylmorphine; however, 7,8-saturated congeners such as dihydromorphine and dihydrocodeine were poor substrates. The enzyme was inactivated by removal of 2-mercaptoethanol from the enzyme solution. The inactivated enzyme was rapidly recovered by the addition of 2-mercaptoethanol. Phenylarsine oxide and CdCl2 (dithiol modifiers) inhibited competitively toward cofactor binding and noncompetitively toward morphine binding. These results suggest that the enzyme possesses the essential thiol groups, probably vicinal dithiol, at or near the cofactor-binding site. Using the partially purified enzyme, 8-(2-hydroxyethylthio)dihydromorphinone was isolated as the product and identified by UV, mass, and NMR spectra. It was confirmed that morphinone proposed as the dehydrogenation product was nonenzymatically and covalently bound to 2-mercaptoethanol. Accordingly, the isolated morphinone-2-mercaptoethanol conjugate must be formed by two steps: enzymatic production of morphinone from morphine and then nonenzymatic binding of 2-mercaptoethanol to morphinone.     

Purification and characterization of a membrane-bound sialidase from pig liver

A membrane-bound sialidase in pig liver microsomes was solubilized with a nonionic detergent, IGEPAL CA630, and purified to homogeneity by sequential chromatographies on SP-Toyopearl, Butyl-Toyopearl (1st), SuperQ-Toyopearl, Hydroxyapatite, Butyl-Toyopearl (2nd), GM1-Cellulofine affinity, and sialic acid-Cellulofine affinity columns. The molecular weight of the purified enzyme was estimated to be 57 kDa on SDS-PAGE. The pH optimum was 4.8 for the activity measured using 4-methylumbelliferyl-alpha-N-acetylneuraminic acid (4MU-Neu5Ac) as the substrate. The enzyme activity was inhibited by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, iodoacetamide and p-chloromercuribenzoic acid. While the enzyme could effectively hydrolyze 4MU-Neu5Ac, it failed to significantly cleave a sialic acid residue(s) from sialyllactose, glycoproteins or gangliosides at pH 4.8. These results suggest that the purified enzyme is a novel sialidase with a substrate specificity distinct from those of known membrane-bound sialidases in mammalian tissues.     

Purification,partial characterization,and subcellular localization of a 38 kilodalton,calcium-regulated protein of Rhizobium fredii USDA208

Calcium is essential for the growth of rhizobia and the formation of nitrogen-fixing root-nodules on legumes, but its precise role in these processes remains unknown. We have found that Rhizobium fredii USDA208 accumulates a major 38 kDa protein when grown in media supplemented with 0.3–2 mM CaCl₂. We have purified this protein and raised polyclonal antibodies against it. The protein initially is synthesized as a 40 kDa precursor which subsequently undergoes calcium-dependent processing to give rise to the mature polypeptide. Subcellular and immunocytochemical localization studies indicate that the 38 kDa protein accumulates preferentially in the periplasmic space. Its N-terminal sequence, AETIKIGVAGPMTG, shows significant homology to the N-termini of amino acid binding proteins from the periplasm, including leucine-, isoleucine-, and valine-specific binding proteins of Pseudomonas aeruginosa and Escherichia coli and a leucine-specific binding protein of E. coli. The R. fredii protein does not, however, bind [³H]-leucine. The 38 kDa protein is encoded by the bacterial chromosome. It is absent in several rhizobia other than R. fredii, but antigenically related polypeptides are present in Escherichia coli and Erwinia carotovora subsp. carotovora.     

Purification and characterization of two components of acid alpha-glucosidase from pig liver

Acid alpha-glucosidase [EC 3.2.1.3] was purified from pig liver by a procedure including Sephadex G-100 affinity chromatography. Electrophoresis on SDS-polyacrylamide gel of the purified enzyme indicated the presence of two components with molecular weights of 73K and 64K. The two components of the enzyme were completely separated, in reasonable yield, by chromatography on a DEAE-5PW column. Both components catalyzed the hydrolysis of the alpha-1,4 and alpha-1,6 linkages of glycogen, maltose, isomaltose, dextrin, and a synthetic glucoside at acid pH. The pH optima of both components were 4.3 for maltase and glucoamylase, and 4.8 for isomaltase and dextrinase. But as to the activity on 4MU-alpha-Glc, the pH optimum of the larger component was 4.8 and that of the smaller component 5.3. The Km values of both components for 4MU-alpha-Glc, maltose, glycogen, isomaltose, and dextrin were 1.0 X 10(-4) M, 9.1 X 10(-3) M, 16.7 mg/ml, 6.7 X 10(-2) M, and 12.5 mg/ml, respectively. Erythritol, Tris, and turanose inhibited the two components competitively. The Ki values of the larger component were 5.0 X 10(-2) M, 13.3 X 10(-3) M, and 3.2 X 10(-3) M, and those of the smaller component were 2.5 X 10(-2) M, 6.1 X 10(-3) M, and 4.7 X 10(-3) M, for erythritol, Tris, and turanose, respectively.     

Purification, characterization, and localization of linamarase in cassava

We have purified cassava (Manihot esculenta) linamarase to apparent homogeneity using a simplified extraction procedure using low pH phosphate buffer. Three isozymes of cassava linamarase were identified in leaves based on differences in isoelectric point. The enzyme is capable of hydrolyzing a number of β-glycosides in addition to linamarin. The enzyme is unusually stable and has a temperature optimum of 55°C. Immunogold labeling studies indicate that linamarase is localized in the cell walls of cassava leaf tissue. Since linamarin must cross the cell wall following synthesis in the leaf for transport to the root, it is likely that linamarin must cross the cell wall in a nonhydrolyzable form, possibly as the diglucoside, linustatin. In addition, we have quantified the levels of linamarin and linamarase activity in leaves of cassava varieties which differ in the linamarin content of their roots. We observed no substantial differences in the steady state linamarin content or linamarase activity of leaves from high or low (root) cyanogenic varieties. These results indicate that the steady state levels of linamarin and linamarase in leaves of high and low cyanogenic varieties are not correlated with the varietal differences in the steady state levels of linamarin in roots.     

Purification, characterization, and subcellular localization of an acid phosphatase from black mustard cell-suspension cultures: comparison with phosphoenolpyruvate phosphatase

An acid phosphatase from Brassica nigra (black mustard) leaf petiole cell-suspension cultures has been purified 1633-fold to a final specific activity of 1225 (mumols orthophosphate produced/min)/mg protein and near homogeneity. The native protein was a glycosylated monomer having a molecular mass of 60 kDa and a pI of 4.5. The enzyme displayed a broad pH optimum of about pH 5.6 and was heat stable. The final preparation hydrolyzed a wide variety of phosphate esters. The highest specificity constants were obtained with 3-phosphoglycerate, 2,3-diphosphoglycerate, PPi, and phosphoenolpyruvate (PEP). The enzyme was activated 1.4-fold by 4 mM Mg2+ or Mn2+, but was strongly inhibited by Mo, Pi, F, and several phosphorylated compounds. Subcellular localization experiments revealed that this nonspecific acid phosphatase is probably a secreted enzyme, localized in the cell wall. By contrast, B. nigra PEP phosphatase appeared to be localized in the cell vacuole. Peptide mapping via CNBr fragmentation was employed to investigate the structural relatedness of the two phosphatases. Their respective CNBr cleavage patterns were dissimilar, suggesting that B. nigra acid and PEP phosphatases are distinct polypeptides. Putative metabolic functions of these two phosphatases are discussed in relation to the biochemical adaptations of B. nigra cell-suspension cultures to nutritional phosphate deprivation.     

Purification and characterization of zeta-crystallin/quinone reductase from guinea pig liver.

zeta-Crystallin, a major lens protein of certain mammalian species, has recently been characterized as a novel and active NADPH:quinone oxidoreductase. Here we report the purification of this protein from guinea pig liver by utilizing sequentially: ammonium sulphate precipitation, Blue Sepharose affinity, cation exchange and hydrophobic chromatography steps. This four-step isolation procedure yielded 118-fold purification and a specific activity of 6 U/mg protein when assayed in the presence of 9,10-phenanthrenequinone. Kinetic, immunological and physical properties of this protein have been found to be identical with those of guinea pig lens zeta-crystallin. Western blot analysis using antibodies raised against zeta-crystallin peptides demonstrated the presence of substantial amounts of this protein in human liver homogenates.     

Purification, characterization, and localization of neuropeptides in the cornea

The immunologically detected neuropeptides methionine enkephalin (ME), substance P (SP), beta-endorphin (beta-End), and alpha-melanocyte stimulating hormone (alpha-MSH) were purified from bovine corneal extracts by gradient, followed by isocratic, reversed phase-high performance liquid chromatography (RP-HPLC) and characterized, after both chromatographic steps, by radioimmunoassay (RIA). Immunologically detected ME and SP were purified from canine corneal extracts by gradient RP-HPLC and characterized by RIA. An anatomical study of the bovine cornea separated the cornea into an epithelium-enriched and a stroma-enriched portion. After gradient RP-HPLC, RIA demonstrated that all the ME-like immunoreactivity was located in the corneal epithelium, whereas the SP-like immunoreactivity was distributed between the stroma and epithelium in an approximate two-to-one ratio.     

Tissue and cellular distribution of alpha-L-iduronidase in the pig

We investigated the alpha-L-iduronidase activity of various pig tissues. Furthermore, we examined the tissues using antibody, enzyme immunoassay (EIA), and immunohistochemical methods. The amounts of enzyme measured by the EIA method in the various tissues were proportional to their enzyme activities and also to their immunohistochemical characteristics. The tissues could thus be classified into three groups: a high enzyme activity group composed of the liver, kidney, and spleen; a moderate activity group comprising the lung, lymph nodes, stomach, ileum, colon, and pancreas; and a low activity group consisting of the heart, diaphragm, iliopsoas muscle, cerebrum, cerebellum, and skin. The molecular weight of the enzyme in each tissue did not reveal any heterogeneity, having two components of 70 KD and 62 KD by Western blot analysis. Immunohistochemically, alpha-L-iduronidase was strongly detected in the lysosomal membranes of cells of the mononuclear phagocyte system, epithelial cells of the proximal tubules in the kidney, and some blastic cells, whereas hepatocytes revealed weak positive reactions. The tissue and cellular distribution of the enzyme appeared to have a close relation to tissues that manifest or are affected by alpha-L-iduronidase deficiency.     

Identification, biochemical characterization, and subcellular localization of allantoate amidohydrolases from Arabidopsis and soybean

Allantoate amidohydrolases (AAHs) hydrolize the ureide allantoate to ureidoglycolate, CO(2), and two molecules of ammonium. Allantoate degradation is required to recycle purine-ring nitrogen in all plants. Tropical legumes additionally transport fixed nitrogen via allantoin and allantoate into the shoot, where it serves as a general nitrogen source. AAHs from Arabidopsis (Arabidopsis thaliana; AtAAH) and from soybean (Glycine max; GmAAH) were cloned, expressed in planta as StrepII-tagged variants, and highly purified from leaf extracts. Both proteins form homodimers and release 2 mol ammonium/mol allantoate. Therefore, they can truly be classified as AAHs. The kinetic constants determined and the half-maximal activation by 2 to 3 microm manganese are consistent with allantoate being the in vivo substrate of manganese-loaded AAHs. The enzymes were strongly inhibited by micromolar concentrations of fluoride as well as by borate, and by millimolar concentrations of L-asparagine and L-aspartate but not D-asparagine. L-Asparagine likely functions as competitive inhibitor. An Ataah T-DNA mutant, unable to grow on allantoin as sole nitrogen source, is rescued by the expression of StrepII-tagged variants of AtAAH and GmAAH, demonstrating that both proteins are functional in vivo. Similarly, an allantoinase (aln) mutant is rescued by a tagged AtAln variant. Fluorescent fusion proteins of allantoinase and both AAHs localize to the endoplasmic reticulum after transient expression and in transgenic plants. These findings demonstrate that after the generation of allantoin in the peroxisome, plant purine degradation continues in the endoplasmic reticulum.